Metatranscriptomics of the Hu sheep rumen microbiome reveals novel cellulases

Abstract

Background Cellulosic biomass has great potential as a renewable biofuel resource. Robust, high-performance enzymes are needed to effectively utilize this valuable resource. In this study, metatranscriptomics was used to explore the carbohydrate-active enzymes (CAZymes), especially glycoside hydrolases (GHs), present in the rumen microbiome of Hu sheep. Select CAZymes were experimentally verified and characterized after cloning and expression in E. coli . Results The metatranscriptomes of six Hu sheep rumen microbiomes yielded 42.3 Gbp of quality-checked sequence data that represented in total 2,380,783 unigenes after de novo assembling using Trinity and clustered with CD-HIT-EST. Annotation using the CAZy database revealed that 2.65% of the unigenes encoded GHs, which were assigned to 111 different CAZymes families. Firmicutes (18.7%) and Bacteroidetes (13.8%) were the major phyla to which the unigenes were taxonomically assigned. In total, 14,489 unigenes were annotated to 15 cellulase-containing GH families, with GH3, GH5 and GH9 being the predominant. From these putative cellulase-encoding unigenes, 4225 open reading frames (ORFs) were predicted to contain 2151 potential cellulase catalytic modules. Additionally, 147 ORFs were found to encode proteins that contain carbohydrate-binding modules (CBMs). Heterogeneous expression of 30 candidate cDNAs from the GH5 family in E. coli BL21 showed that 17 of the tested proteins had endoglucanase activity, while 7 exhibited exoglucanase activity. Interestingly, two of the GH5 proteins (Cel5A-h28 and Cel5A-h11) showed high specific activity against carboxymethylcellulose (CMC) and p -nitrophenyl-β- d -cellobioside (pNPC) (222.2 and 142.8 U/mg), respectively. The optimal pH value for activity of Cel5A-h11 and Cel5A-h28 was 6.0 for both enzymes, and optimal temperatures were 40 and 50 °C, respectively. Both enzymes retained over 70 and 60%, respectively, of their original activities after incubation at 40 °C for 60 min. However, their activities were rapidly diminished upon exposure to higher temperatures. Cel5A-h11 and Cel5A-h28 retained more than 80 and 60% of their maximal enzymatic activities after incubation for 16 h in buffered solutions in the pH range from 4.0 to 9.0. Conclusion The metatranscriptomic results revealed that the rumen microbiome of Hu sheep encoded a repertoire of new enzymes capable of cellulose degradation and metatranscriptomics was an effective method to discover novel cellulases for biotechnological applications. Electronic supplementary material The online version of this article (10.1186/s13068-019-1498-4) contains supplementary material, which is available to authorized users.

Metatranscriptomic analysis to define the Secrebiome, and 16S rRNA profiling of the gut microbiome in obesity and metabolic syndrome of Mexican children

“…The N50 of our metatranscriptome assembly was ~ 700 nt, which is congruent to the expected length of Prokaryotic mRNA molecules, (~ 900 pb long). Additionally, > 54% of the total reads from our samples mapped back to the assembly, suggesting an adequate assembly, even better than the ones obtained in other studies using a similar methodology [45]. Indeed, when we remapped sequencing data from different cohorts, we recovered ~ 25% of the reads despite them being of healthy adults from different populations, so this metatranscriptome and Secrebiome could be considered as a reference for further studies in different populations.…”

Ruminal microbiota–host interaction and its effect on nutrient metabolism

“…The application of omics technologies reveals more and more cellulolytic bacteria. Studies using metatranscriptomics found that the rumen microbiome in Hu sheep (a local breed in China) encoded a repertoire of new cellulases ( He et al., 2019 ). Cellulases enable the utilization of low-quality roughage (e.g., corn stover) and can be used to digest roughage for ruminants.…”

“…In addition, animals can use some biomass that cannot be used by humans, which can effectively solve the problem of competition for food between humans and livestock. For example, ruminants can utilize plant cell wall biomass ( Dai et al., 2015 ; He et al., 2019 ), many by-products of agriculture and food industry as feed owing to the rumen microbiota ( Kimball, 1991 ; Westendorf, 2000 ; Malaweera and Wijesundara, 2014 ). It has been suggested that ruminant livestock farming has the potential to minimize the use of human-edible grains by utilizing available forage resources within a given system ( Eisler et al., 2014 ).…”

Innovations in CAZyme gene diversity and its modification for biorefinery applications

“…It led to breakthrough of the first exo-1,4-β-xylanase (act on xylo-oligomers) from family 11 of glycoside hydrolase (GH11) [ 54 ]. Using approaches of RT-PCR, the microbial origin and expression level of cellulase gene was examined in the rumen of Hu sheep [ 55 ]. Metatranscriptomic analysis of rumen microorganisms of adult Holstein cows indicated high quality of non-rRNA reads and the transcripts encoding GHs and some carbohydrate binding molecules [ 56 ].…”

“…These techniques have already showed their efficiency by being able to discover various new non culturable bacteria possessing polysaccharide degrading abilities [ [195] , [196] , [197] ] as well as novel CAZymes [ 55 , 198 , 199 ] with various roles in polysaccharides degradation abilities which would not have been possible with the classical culture-based methods [ 200 ]. Metagenomic study of rumen of Mehsani buffalo and Korean black goat rumen unearthed novel GH26 and KG51 genes respectively which were than expressed and characterized [ 201 , 202 ].…”